The neural circuits governing behaviors vital to mammals, such as locomotion and respiration, rely on the ability of motor neurons (MNs) to establish selective connections with target cells both centrally and peripherally. Motor neurons innervating specific muscle targets are specified by the large family of chromosomally arrayed Hox transcription factors. A key aspect of Hox gene function is to segregate motor neurons into topographically organized columnar and pool subtypes. While it has been suggested the somatotopic organization of MNs evolved to facilitate the activation of an increasingly more complex limb musculature, it is largely unknown how MNs cluster into columns, and what role MN position plays in shaping the specificity of connections within motor networks. In this proposal we will investigate the function of Pbx genes, essential co-factors of Hox proteins, in the formation of MN topographic maps and in the development of motor circuits. The major goals of this proposal are to: 1) to assess the role of Pbx genes in the organization and connectivity of spinal motor neurons, 2) to determine the mechanisms through which motor neurons are topographically organized, and 3) to define the role of MN position and identity in spinal circuit assembly. In Aim1 we will define the function of Pbx genes in MN differentiation using genetic manipulations and histological assays. In Aim2 we will identity the targets of Pbx proteins in MNs, and assess their function and mechanisms of regulation. In Aim3 we will assess how motor neuron position and identity influences the specificity of connections with presynaptic interneuron populations. By building off our in depth knowledge of motor neuron specification programs in mammals, these studies should provide basic insights into the mechanisms through which motor circuits are assembled.